BACKGROUND AND OBJECTIVE: DNA damage-induced chromatin reorganization is emerging to be a key aspect of eukaryotic DNA repair. Much has been learned about the role of histone modifications as landing pads for repair effectors, thereby modulating or directing their recruitment to sites of damage. Recent work suggests that structural changes in the break-surrounding chromatin environment may be equally important in directing the repair process, with implications ranging from repair factor accessibility to break-proximal transcriptional silencing. In light of this complexity, we decided to take an unbiased approach to dissect the role of chromatin in DNA repair, identify its most critical components and explore their functional relevance. RESULTS AND FUTURE DIRECTIONS: To gain insight into the role of chromatin in DSB repair, we performed RNAi-based high-throughput screening of a comprehensive list of 400 Gene Ontology-annotated chromatin modifiers. Repair efficiency was determined using the previously established, U2OS cell-based DR-GFP reporter system, in which DNA repair by homologous recombination (HR) results in restoration of a functional GFP gene and HR efficiency can, thus, be measured as the fraction of GFP+ cells. We found a large number of chromatin-modifying enzymes to be involved in DNA break repair. Specifically, we identified two repressive chromatin components, the macro-histone variant macroH2A1 and the H3K9 methyltransferase and tumor suppressor PRDM2, which together modulate the choice between the antagonistic DSB repair mediators BRCA1 and 53BP1. The macroH2A1/PRDM2 module mediates an unexpected shift from accessible to condensed chromatin that requires the ATM-dependent accumulation of both proteins at DSBs to promote DSB-flanking H3K9-dimethylation. Remarkably, loss of macroH2A1 or PRDM2 as well as experimentally induced chromatin decondensation impairs BRCA1, but not 53BP1 retention at DSBs. As a result, macroH2A1 and/or PRDM2 depletion causes epistatic defects in DSB end resection, homology-directed repair and the resistance to PARP inhibition - all hallmarks of BRCA1-deficient tumors. Together, these findings identify dynamic, DSB-associated chromatin reorganization as a critical modulator of BRCA1-dependent genome maintenance. We are currently investigating the consequences of macroH2A1-associated repressive chromatin formation during replication stress, which involves HR-dependent genome maintenance pathways and has been linked to cellular dysfunction and, ultimately, senescence. We further initiated a new aim to investigate the role of other HR repair-associated chromatin modifiers identified in our screen. Given recent insight into the importance of tightly controlled chromatin and repair factor ubiquitination, our current efforts focus on the USP-family deubiquitinating enzyme USP21. We found that USP21 is a specific regulator of HR that is recruited to sites of DNA damage, where it counteracts the formation of ubiquitin chains. Consistent with previous reports, we identified histone H2A as one of its targets. To explain the HR-specific defect of USP21-deficient cells, and we next performed a detailed dissection of repair factor recruitment in the presence of absence of USP21. Remarkably, USP21 ablation resulted in a specific loss of Rad51 foci at sites of damage, despite intact recruitment of upstream effectors. Biochemical analyses identified USP21 as a novel regulator of the BRCA2/Rad51 complex. Moreover, we found that USP21 expression is increased in hepatocellular carcinoma (HCC) and correlates with poor survival outcome. Tumor cells are rapidly dividing and, hence, prone to the accumulation of replication-associated DSBs, resulting in their dependence on efficient HR. Consistent with this, increased Rad51 expression shows a survival defect similar to USP21 in human HCC cohorts. Our findings, therefore, point to a role for USP21 in promoting tumor cell survival by ensuring BRCA2/Rad51 stability and, thus, resistance to genotoxic stress. To test this hypothesis, we are currently using USP21 knockdown or overexpression in HCC cell lines, as well as xenograft experiments to determine BRCA2/Rad51 stability, repair efficiency and tumor cell survival. Finally, we are analyzing the impact of USP21 loss on genome maintenance in mice using a previously established USP21 knockout mouse model. Together, this aim is expected to shed light on a novel aspect of ubiquitin-dependent DSB repair with the potential to modulate tumor cell survival. IMPLICATIONS: Defects in HR promote genomic instability, which can result in malignant transformation. However, once transformation has occurred, DSB repair, and HR in particular are essential for the survival of rapidly dividing tumor cells. Determining the factors that control HR is, thus, critical for our understanding of DSB repair in general and tumorigenesis in particular. Together, our work thus far has demonstrated that, by elucidating the function of (chromatin-associated) HR mediators, we can gain important mechanistic insight into HR repair aspects with implications for the targeted manipulation of repair outcome and possibly tumor initiation and/or progression.